Abstract
Objective. To investigate the effects of cerium dioxide (CeO2) nanoparticles on the inflammatory response of peritonitis rats induced by Staphylococcus epidermidis infection. Methods. Green tea polyphenol CeO2 nanoparticles were synthesized and characterized by transmission microscopy, ultraviolet-visible spectroscopy, FT-IR, and powder diffractometer. 40 male adult SD rats were randomly divided into 4 groups (n = 10 each): a control group, a model group, a CeO2 group, and a CeO2 + model group. Staphylococcus epidermidis solution was injected intraperitoneally with 107 CFU/ml of bacterial solution in the model group, while the control group was injected intraperitoneally with the same amount of normal saline, and the CeO2 and CeO2 + model groups were injected with 0.5 mg/kg CeO2 nanoparticles through the tail vein for 2 h and then injected with saline or bacterial solution for 2 h, respectively. After 0 h, 3 h, 12 h, 24 h, and 48 h of model construction, rats were sacrificed, and serum and peritoneal lavage fluid were collected. The total number of leukocytes and the percentage of each type of leukocytes in the peritoneal lavage fluid were determined. Enzyme-linked immunosorbent assay (ELISA) was used to detect the level of inflammatory factor TNF-α in serum and peritoneal lavage fluid, and myeloperoxidase (MPO) activity in peritoneal tissue was also measured. In addition, real-time fluorescence quantitative PCR (RT-PCR) was used to measure the expression of TLR2 and TLR4 in peritoneal tissue, and western blotting was used to detect the expression of TLR2, TLR4, and the activation of NF-κB signaling pathways as well. Results. The CeO2 has an average size of 37 ± 3 nm with binding activity to proteins, phenolic compounds, and alkaloids. After counting the white blood cells in the peritoneal lavage fluid, it was found that the total number of white blood cells and the percentage of neutrophils in the model group were significantly increased (both ), and CeO2 treatment significantly reversed the above changes (both ). The ELISA results showed that compared with the control group, the TNF-α in the peritoneal lavage fluid and serum of the model group increased in a time-dependent manner (all ); however, there was no significant change in the CeO2 group (); at the same time in the CeO2 + model group, the TNF-α content was significantly reduced (all ). Detection of MPO activity in peritoneal tissue revealed that MPO activity was significantly increased under peritonitis (all ), and CeO2 treatment could mitigate that increase (all ). RT-PCR results showed that compared with the control group, the expression of TLR2 and TLR4 mRNA levels in the peritoneum of the model group were increased in a time-dependent manner (all ), and there was no significant change in the CeO2 group (); however, TLR2 and TLR4 mRNA levels were significantly reduced in the CeO2 + model group (all ). Western blotting test was performed on the peritoneal tissue collected after 48 h of the model establishment. Compared with the control group, the levels of TLR2, TLR4, p–NF–κB, and p-IκBα protein in the model group were significantly increased (all ), while CeO2 group showed no significant changes () and administration of CeO2 before model construction can significantly reverse the above protein activation (all ). Conclusion. CeO2 nanoparticles have anti-inflammatory effects in peritonitis caused by Staphylococcus epidermidis infection.
1. Background
Although the diagnosis and treatment of diseases have progressed with the development of medicine, the incidence of deaths associated with celiac infection and severe peritonitis remains high. Peritonitis can occur when infectious pathogens/microorganisms or their toxic metabolites are present in the peritoneum [1]. If the infection is aggravated due to improper handling and other reasons, it often develops into diffuse peritonitis and organ dysfunction. Peritoneal dialysis is also an important cause of peritonitis. It is often referred to as peritoneal dialysis-related peritonitis and among which, Staphylococcus epidermidis infection is the most common type [2]. It is reported that most of the organ damage in peritonitis is caused by the development of systemic inflammatory response syndrome (SIRS). In the occurrence and development of SIRS, there is an increase in inflammatory cytokines, tissue reactive oxygen levels, and an increase of monocytes, macrophages, and neutrophils infiltration in different organs; all of these combined together can cause tissue damage [3].
Cerium is a lanthanide series metal that can undergo a redox cycle from Ce4+ (oxidation) to Ce3+ (reduction) state and often exists in the form of cerium dioxide (CeO2). Previous studies have confirmed that CeO2 nanoparticles can act as superoxide dismutase mimics [4] and catalase mimics [5] and can reduce pulmonary hypertension [6] and reduce hypoxia-induced oxidative stress [7] as well as inhibiting cyclophosphamide-induced apoptosis [8]. In the study of peritonitis, CeO2 nanoparticles have been reported to reduce peritonitis-related SIRS [2], and other studies have found that CeO2 nanoparticles can improve diaphragmatic dysfunction caused by peritonitis [9]. In addition, green tea polyphenols, as the main ingredients of antioxidants, are well known for their antioxidant, anti-inflammatory, anticancer, anticardiolipin, antimicrobial, antihyperglycemic, and antiobesity properties [10].
In this study, a rat model of Staphylococcus epidermidis-induced peritonitis was established to investigate the effects of biosynthetic CeO2 nanoparticles on the inflammation of this model.
2. Materials and Methods
2.1. Plant Collection and Extraction
Green tea leaves were collected and dried to make a powder. 10 g of dried tea powder was added to a 250 ml beaker, and 100 ml of distilled water was added for continuous boiling for 10 min. The boiling solution was filtered through filter paper, and the filtrate was collected and stored at 4°C.
2.2. Synthesis of Polyphenol CeO2 Nanoparticles
20 ml of the above filtrate was added to the conical flask, heated to boiling, and when it was cooled to 50°C, ammonium cerium nitrate solution (1 g in 50 ml of distilled water) was mixed with the filtrate and stirred until the solution turned to milky yellow. The solution was then transferred to a clean centrifuge tube and was centrifuged (12,000 rpm/min) at 4°C for 10 minutes. The supernatant was then discarded, and the milky solid precipitate at the bottom of the centrifuge tube was transferred to a quartz crucible and placed in a muffle furnace (calcined at 600°C for 1 h). A yellow-white solid was obtained after 1 h, and CeO2 nanoparticles were obtained by further grinding [11].
2.3. Characterization of Synthetic CeO2 Nanoparticles
The synthesized CeO2 nanoparticles were observed using a transmission microscope (JEM-2010, JEOL, Japan) and characterized and identified by UV-visible spectroscopy (125 spectrophotometers, PerkinElmer, Germany), and an FT-IR instrument (JASCO, USA) was used for infrared spectrum analysis. In addition, a powder diffractometer (D8, BRUKER-AXS, Germany) was used to analyze the XRD pattern.
2.4. Experimental Design and Establishment of the Rat Model of Peritonitis
40 male Sprague Dawley rats (10-week-old, 250–300 g) were purchased from Changzhou Cavins Experimental Animal Co., Ltd. The rats were housed in a cage with an indoor temperature of 20°C–24°C and alternating light and dark cycles (12 h each) within 2 weeks of the experiment. The rats had free access to standard rodent food and water. This study was reviewed and approved by the ethics committee of our hospital, and all surgical procedures were performed in accordance with the guidelines. The rats were randomly divided into 4 groups (n = 10 each): the control group, the model group, the CeO2 group, and the CeO2 + model group. For the preparation of Staphylococcus epidermidis solution, please refer to the related literature [12]. Specifically, Staphylococcus epidermidis strain (ATCC 35984) was inoculated into 300 ml of conventional culture medium and shaken at 250 rpm/min at 37 °C overnight. The next day, it was centrifuged at 10,000 rpm/min for 5 minutes at 4°C. The supernatant was discarded and the pellet was collected. The pellet was then suspended in 50 ml of prechilled normal saline, centrifuged, and washed at 5000 rpm/min for 5 minutes, which was repeated three times. The final pellet was then resuspended in prechilled normal saline to prepare Staphylococcus epidermidis solution with a concentration of 107 CFU/ml. The solution was stored at 4°C for subsequent experiments. Rats in the control group were injected intraperitoneally with saline; model group was injected intraperitoneally with Staphylococcus epidermidis solution 107 CFU/ml; CeO2 group was injected with 0.5 mg/kg CeO2 nanoparticles (dissolved in 200 ml sterilized water) via tail vein 2 h before intraperitoneal saline injection; CeO2 + model group was intraperitoneally injected with 0.5 mg/kg CeO2 nanoparticles as well (dissolved in 200 ml of sterilized water) via tail vein 2 h before model construction and Staphylococcus epidermidis solution injection. Rats were then sacrificed at 0 h, 3 h, 12 h, 24 h, and 48 h after model construction and whole blood was centrifuged to obtain serum, peritoneal lavage fluid, and peritoneal tissue for the subsequent experiment.
2.5. Counting the Number of White Blood Cells in the Peritoneal Lavage Fluid
The peritoneal lavage fluid was centrifuged at 1,000 rpm for 10 min at room temperature, and the supernatant was collected and stored at−80°C for subsequent detection of inflammatory factors. The pellet was collected and resuspended in 200 μl PBS containing 0.6 mM EDTA. Density gradient centrifugation and Wright-Giemsa staining were used to count the white blood cells.
2.6. Detection of Inflammatory Factors in Serum and Peritoneal Lavage Fluid by ELISA
The concentration of TNF-α in peritoneal lavage fluid and serum was determined using the ELISA kit (R&D, USA) according to the manufacturer’s instructions.
2.7. Detection of Myeloperoxidase Levels in Peritoneal Tissue
3 ml of 0.05 M K-phosphate buffer (pH = 6.0) containing 0.5% cetyltrimethylammonium bromide was added to a total of 200 mg peritoneal sample and sonicated for 20 s to prepare a homogenate. Homogenate was then freeze-thawed for 3 times and centrifuged at 10,000 rpm for 15 min. Then, 0.1 ml supernatant was added and mixed to a 2.9 ml 0.05 M K-phosphate buffer (pH = 6.0) containing 0.53 mM o-diphenylamine and 0.15 mM H2O2. A spectrophotometer was used to measure the solution absorbance at 460 nm every 15 s continuously for 5 min. The MPO activity is expressed in U (units)/mg protein, and 1 U is defined as the degradation of 1 μM peroxide/min at 25 °C [13].
2.8. TLR2 and TLR4 mRNA Levels in Peritoneal Tissue Detected by Real-Time Fluorescence Quantitative PCR (RT-PCR)
Total RNA was extracted from peritoneal tissue using an RNA extraction kit (DP420, Tiangen, China), and the concentration and purity were determined by a spectrophotometer. 1 μg of RNA was reverse transcribed into cDNA, followed by SYBR Green real-time fluorescence quantitative amplification. The primer sequences are shown in Table 1. β-Actin was used as the internal reference gene, and the relative expression of the target gene was calculated using the 2−△△Ct method.
2.9. Expression of TLR2, TLR4, and NF-κb Signaling Pathway Proteins in Peritoneal Tissue Determined by Western Blotting
After washing the tissue with prechilled PBS, a certain amount of RIPA strong lysate and protease inhibitor (both purchased from Biyuntian, China) was added, and the tissue was completely lysed under ultrasound. The total protein from the peritoneal tissue was then extracted and the BCA protein concentration determination kit (Biyuntian, China) was used for protein quantification before protein was heated to denature. A certain amount of protein was added to the SDS-PAGE gel wells for electrophoresis before transferring to membranes. The membranes were then blocked, and primary antibodies were added. After incubation, membrane washing, secondary antibody incubation, membrane washing, and other steps, ECL luminescent solution was dropped on the membrane for fluorescence development. TLR2, TLR4, NF-κB p65, phosphorylated NF-κB p65 (p–NF–κB p65), IκBα, p-IκBα, and β-actin were purchased from Cell Signaling Technology, USA.
2.10. Statistical Analysis
Measurement data in this study were expressed in the form of mean ± standard deviation. t test was used for comparisons between the two groups and ANOVA analysis was used for comparison within groups under univariate conditions. was considered statistically significant.
3. Results
3.1. Transmission Electron Microscopy and UV-Visible Spectrum Analysis of CeO2 Nanoparticles
The transmission electron microscope image of CeO2 nanoparticles is shown in Figure 1(a). The UV-visible spectrum analysis of polyphenol CeO2 nanoparticle mixtures was performed every 30 minutes. As shown in Figure 1(b), the surface plasmon resonance peak observed at 267 nm indicates the formation of CeO2 nanoparticles.
(a)
(b)
3.2. FT-IR Analysis of CeO2 Nanoparticles
As shown in Figure 2, the functional group wavelengths of polyphenol CeO2 nanoparticles were from 400 cm−1 to 4000 cm−1. And at 3423 cm−1 (COOH–OH), 2960 cm−1 (CH), 1618 cm−1 (amide II band or N-diamine), 1421 cm−1 (COH), 1270 cm−1 and 1067 cm−1 (thio and vinyl), 916 cm−1, and 831 cm−1 (organic sulfur compounds), CeO2 reached the peak, confirming the existence of multiple functional groups in tea polyphenols. FT-IR analysis of CeO2 nanoparticles synthesized by polyphenols showed that CeO2 nanoparticles can be combined with compounds such as proteins, phenolic compounds, and alkaloids. These chemicals play a capping reagent role in the synthesis of nanoparticles.
3.3. XRD Analysis of CeO2 Nanoparticles
As shown in Figure 3, the intensity of XRD was recorded from 20° to 80° at two angles. All the peaks obtained from the diffraction pattern were well characterized by CeO2 nanoparticles. The intensities of the narrow-tip diffraction peaks appearing at 2θ were 28.02, 33.08, 47.01, 57.45, 59.37, 69.39, and 78.43, corresponding to the lattice planes 111, 200, 220, 311, 222, 400, and 331 of CeO2 nanoparticles. The above results showed that CeO2 nanoparticles are spherical. The Debye–Scherrer equation was adopted to calculate the average size of the nanoparticles that was 37 ± 3 nm [13].
3.4. Number of Leukocytes in Peritoneal Lavage Fluid of Rats in Each Group
As shown in Figure 4(a), compared with the control group, the total number of white blood cells in the peritoneal lavage fluid of the model group was significantly increased (all ) and was time-dependent (all ); however, there was no significant difference in the CeO2 group (), whereas compared with the model group as shown in Figure 4(b), 4(c), and 4(d), pretreatment with CeO2 significantly reduced the total number of white blood cells (all ). The changes in the percentage of neutrophils in each group were consistent with the changes in the total number of white blood cells, while the percentage changes in macrophages and lymphocytes were the opposite.
(a)
(b)
(c)
(d)
3.5. Expression of Inflammatory Factors in Peritoneal Lavage Fluid and Serum of Rats in Each Group
As shown in Table 2, compared with the control group, TNF-α levels in the peritoneal lavage fluid of the model group rats were increased in a time-dependent manner during the period of 3–48 h after Staphylococcus epidermidis stimulation (); however, compared with the control group, there was no significant difference in CeO2 group (). On the other hand, compared with the model group, the administration of CeO2 before infection significantly reduced TNF-α levels (). Table 3 shows that the serum results were consistent with the results obtained from peritoneal lavage fluid.
3.6. MPO Activity in Peritoneal Tissue of Rats in Each Group
As shown in Figure 5, compared with the control group, the MPO activity in the peritoneal tissue of the model group was significantly higher (all ) and was time-dependent (all ). No significant difference in the CeO2 group () was observed compared to the control group. However, compared with the model group, the administration of CeO2 before infection significantly reduced MPO activity (all ).
3.7. mRNA Levels of TLR2 and TLR4 in the Peritoneal Tissue of Rats in Each Group
As shown in Figure 6, RT-PCR results showed that compared with the control group, mRNA expression levels of TLR2 and TLR4 in the peritoneal tissue of the model group rats increased gradually after infection with Staphylococcus epidermidis (all ), but there was no significant difference in CeO2 group (). However, compared with the model group, mRNA expression levels of TLR2 (Figure 6(a)) and TLR4 (Figure 6(b)) in the CeO2 + model group were reduced, and the difference was significant (all ). ELISA and RT-PCR results suggested that with the establishment of the model, the inflammatory response gradually increased with time. Therefore, in the western blotting experiment, 48 h was chosen as the final detection time point.
(a)
(b)
3.8. Expression of TLR2, TLR4, and NF-κB Signaling Pathway Proteins in the Peritoneal Tissue of Each Group of Rats
As shown in Figure 7, western blot results showed that compared with the control group, the levels of TLR2, TLR4, p–NF–κB, and p-IκBα protein in the model group were significantly increased (all ). However, no significant changes were found in the CeO2 group (). Compared with the model group, the levels of TLR2, TLR4, p–NF–κB, and p-IκBα protein in the CeO2 + model group were significantly reduced (all ).
4. Discussion
Peritonitis is a major complication of peritoneal dialysis, a technique used to treat end-stage renal disease [14]. Among others, Staphylococcus epidermidis is an important pathogenic bacterium in the peritonitis with extremely strong drug resistance, so it is very difficult to completely remove them. In the occurrence of peritonitis, pathogenic bacteria can enter the abdominal cavity through the catheter or catheter outlet, activating peritoneal mesothelial cells and macrophages, which in turn releases a series of products including pro-inflammatory mediators to cause an inflammatory response [15].
Nanotechnology has shown great potential in improving the quality of daily life and has been widely used in the fields of new material production, industry, consumer goods, and medical applications. Studies have found the positive effects of CeO2 nanoparticles in antioxidant therapy, neuroprotection, radiation protection, and eye protection [16]. Meldrum et al. have confirmed that CeO2 nanoparticles can affect the occurrence of allergic airway diseases by regulating the pulmonary inflammation caused by exhaust particles and allergens [17]. With the in-depth study of nanomedicine, the organic combination of nanoparticles and bioactive drugs has gradually become a research hotspot. Green tea polyphenols, as the main component of antioxidants, have anti-inflammatory properties [10]. In view of the anti-inflammatory advantages of CeO2 nanoparticles, and no related reports on the effects of CeO2 nanoparticles on peritonitis, in this study, CeO2 nanoparticles were first biosynthesized based on the literature, and the nanoparticles were further characterized and identified by transmission microscope observation, ultraviolet-visible spectrum, infrared spectrum, and XRD pattern and then peritonitis induced by Staphylococcus epidermidis in rats was established to explore the role of biosynthesized CeO2 nanoparticles on the inflammatory response to peritonitis.
The NF-κB pathway is a typical proinflammatory signaling pathway, which plays an important role in the pathogenesis of inflammatory reactive diseases. NF-κB can be activated through the activation of Toll-like receptors (TLRs) to upregulate proinflammatory genes including cytokines, chemokines, and adhesion molecules [18]. TLRs are single, type I transmembrane glycoprotein that plays an important defensive role by activating immune cell responses in both innate and adaptive immune responses [19]. When the ligand binds to TLRs, it can trigger the myeloid differentiation primary response protein 88-TNFα receptor-related factor 6-transforming growth factor β-activated kinase 1-IκB kinase protein phosphorylation, leading to activation of NF-κB light chain enhancer, thereby promoting the production of inflammatory factors and inducing an inflammatory response. In this study, by detecting the mRNA and protein expression levels of TLR2 and TLR4, we found that the expression of the two receptors increased to varying degrees under the condition of peritonitis. By further testing the downstream IκBα and NF-κB signaling pathways, we found their phosphorylation levels were increased, suggesting that this signaling pathway was activated. In addition, the number of white blood cells in the peritoneal lavage fluid and the percentage of various types of white blood cells, the activity of myeloperoxidase, and the protein expression level of the inflammatory factor TNF-α were also measured in this study. The white blood cell count and the proportion of neutrophils can directly reveal the inflammatory status of the cells; MPO is a heme protein synthesized during the myeloid differentiation process. As a special component in neutrophils, its concentration is related to the severity of inflammation; TNF-α is a proinflammatory factor secreted by monocytes and is known for its strong inflammatory effect. This study tested the above indicators to confirm that the peritonitis status can increase the total number of white blood cells and the proportion of neutrophils, enhance MPO activity, and induce a significant increase in TNF-α levels in peritoneal lavage fluid and serum. And CeO2 nanoparticles can reduce the levels of the above indicators to different degrees and thus exert the anti-inflammatory effects.
In summary, this study found that in the occurrence and development of peritonitis caused by Staphylococcus epidermidis, CeO2 inhibited the transcription of TLR2 and TLR4 genes, inhibited the activation of the NF-κB signaling pathway, and further inhibited its downstream inflammatory factor TNF-α and reduced the number of white blood cells, thereby mitigating the occurrence of inflammatory reactions. Therefore, it is suggested that CeO2 plays a protective role in Staphylococcus epidermidis-induced peritonitis and provides new ideas for the treatment of this disease.
Data Availability
The data used to support the findings of this study are included within the article.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Acknowledgments
This study was supported by grant NSFC81300346 from the National Natural Science Foundation of China.